Isolated mononuclear cells were first labeled with Zombie® NIR stain (BioLegend) according to manufacturer’s instructions to discriminate live/dead cells. MCs were then labeled with flourochrome-conjugated monoclonal antibodies, listed in Table 2 , as previously described 17 . Samples were then acquired using the LSR Fortessa in a five laser (355nm, 405nm, 488nm, 562nm, 633nm) 20-detector configuration (BD Biosciences).
Zombie nir stain
Manufactured by BioLegend
Zombie® NIR stain is a reagent used for the detection and quantification of dead cells in flow cytometry applications. It binds to cells with compromised membranes, allowing for the identification of non-viable cells in a sample.
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Lab products found in correlation
2 protocols using zombie nir stain
1
Detailed Immunophenotyping of Mononuclear Cells
2
Flow Cytometry Immunophenotyping Protocol
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Samples were re-suspended in FACS buffer (PBS containing 2% FBS (Gibco)) + 1:1,000 Zombie NIR stain (BioLegend) and plated onto 96-well round-bottomed plates. Samples were incubated at 4 °C for 30 min, then pelleted and re-suspended in 200 µl of 1:400 panel antibodies + TruStain FcX (BioLegend). Plates were incubated at 4 °C for at least 40 min. After pelleting, samples were re-suspended in 200 µl 4% Pierce 16% formaldehyde (w/v) (Life Technologies) and incubated at room temperature for 10 min in the dark. Samples were pelleted and re-suspended in FACS buffer, sealed in parafilm and stored at 4 °C until analyzed.
To run the samples, each well was transferred into 1.2 ml round-bottom tubes (Stellar Scientific) and run on the BD LSRFortessa (BD Biosciences). Samples were analyzed using FlowJo (v.10.9.0).
To run the samples, each well was transferred into 1.2 ml round-bottom tubes (Stellar Scientific) and run on the BD LSRFortessa (BD Biosciences). Samples were analyzed using FlowJo (v.10.9.0).
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